Innovative chimeric antigen receptor (CAR) T cell designs and combinational approaches are needed for enhancing therapeutic effectiveness in solid tumors. We developed and assessed a novel dual-targeting CAR-T therapy that combines an αPDL1.CD28 chimeric receptor with a second-generation αCD133 CAR to target CD133+ tumors. The αPDL1.CD28 structure activated the CD3ζ signaling in cis by clustering with αCD133 CAR via CD28 dimerization. Binding to programmed cell death ligand-1 (PD-L1) through αPD-L1 CAR improved the CD133-targeted cytotoxic function of T cells by enhancing activation signals and countering inhibitory signals. Combination with programmed cell death receptor-1 (PD-1) blockade further disrupted the PD-L1/PD-1 inhibitory signal, achieving prolonged therapeutic efficacy. Moreover, radiation pre-conditioning (10 Gy/1 fraction or 4 Gy/2 fractions) maximized the antitumor effects of CAR-T plus PD-1 blockade, inducing complete tumor regression in mice. Radiation induced a unique tissue-resident memory CAR-T cell phenotype with high CXCR6 and CD103 expression. As the ligand of CD103, E-cadherin expression increased in tumor cells after irradiation, potentially mediating E-cadherin-CD103 interactions between tumor cells and tissue-resident memory T cells. Our study introduces a novel dual-targeting CD133/PD-L1 CAR-T cell and further demonstrates the efficacy and rationale of the triple-combination approach in solid tumors.

Although immune checkpoint inhibitors have changed the treatment paradigm for non-small cell lung cancer (NSCLC), not all patients benefit from them. Therefore, there is an urgent need to explore novel immune checkpoint inhibitors. Neuropilin-1 (Nrp-1) is a unique immune checkpoint capable of exerting antitumor effects through CD8+ T cells. It is also a T-cell memory checkpoint that regulates long-term antitumor immunity. However, its role in NSCLC remains unclear. The aim of this study was to develop a fully human anti-Nrp-1 antibody with therapeutic effects against NSCLC in vitro and in vivo. We screened and constructed of a high-affinity anti-Nrp-1 IgG antibody from a constructed high-capacity fully human single-chain fragment variable (scFv) phage library. This novel anti-Nrp-1 IgG antibody partially restored the killing function of exhausted CD8+ T cells in malignant pleural fluid in vitro. Co-culture of peripheral blood mononuclear cells (PBMC) with A549 and the addition of anti-Nrp1-IgG enhanced the killing of A549 target cells, leading to an increase in late-stage apoptosis of target cells. Importantly, anti-Nrp1-IgG treatment significantly reduced tumor volume in a mouse model of lung cancer with humanized immune system. These findings suggest that 53-IgG has a promising application as a potent Nrp-1-targeting agent in NSCLC immunotherapy.

Pharmacological agents regarding the most optimal treatments of acute pancreatitis remain. One-carbon metabolism nutrients as therapeutic agents in many diseases might be involved in acute pancreatitis. The roles are acquired exploration in acute pancreatitis. We utilized Mendelian randomization to assess the causal impact of folate, homocysteine, and vitamin B12 (VB12) on acute pancreatitis. Wild-type and corresponding genetically modified mouse models were used to verify the genetic correlating findings. A negative association between genetically predicted serum VB12 levels and risks of acute pancreatitis was identified in human population. The transcobalamin receptor (TCblR)/CD320 gene ablation that decreased cellular VB12 uptake and ATP production in pancreatic tissues promoted necrosis, resulting in much severe pathological changes of induced acute pancreatitis in mice. VB12 pretreatment and posttreatment dramatically increased ATP levels in pancreatic tissues and reduced the necrosis, then the elevated levels of amylase in serum, the levels of CK-19, the activity of trypsin, and T lymphocyte infiltration in pancreatic tissues, prevented the pancreatic gross loss and ameliorated histopathological changes of mouse pancreases with induced acute pancreatitis. The results reveal that VB12 is potential as a therapeutic agent to inhibit tissue injuries and adaptive inflammatory responses in the pancreas in patients with acute pancreatitis.

BACKGROUND:Immune checkpoint inhibitors (ICIs) provide modest but unsatisfactory benefits for extensive-stage small cell lung cancer (ES-SCLC). Developing strategies for treating ES-SCLC is critical. METHODS:We preliminarily explored the outcomes of salvage low-dose radiotherapy (LDRT) plus ICI on refractory SCLC patients. Next, we evaluated the combinational efficacy in murine SCLC. The tumor immune microenvironment (TIME) was analyzed for mechanistic study. Subsequently, we conducted a multicenter, prospective phase II trial that administered concurrent thoracic LDRT plus chemoimmunotherapy to treatment-naive ES-SCLC patients (MATCH trial, NCT04622228). The primary endpoint was confirmed objective response rate (ORR), and the key secondary endpoints included progression-free survival (PFS) and safety. FINDINGS:Fifteen refractory SCLC patients treated with LDRT plus ICI were retrospectively reviewed. The ORR was 73.3% (95% confidence interval [CI], 44.9-92.2). We identified a specific dose of LDRT (15 Gy/5 fractions) that exhibited growth retardation and improved survival in murine SCLC when combined with ICIs. This combination recruited a special T cell population, TCF1+ PD-1+ CD8+ stem-like T cells, from tumor-draining lymph nodes into the TIME. The MATCH trial showed a confirmed ORR of 87.5% (95% CI, 75.9-94.8). The median PFS was 6.9 months (95% CI, 5.4-9.3). CONCLUSIONS:These findings verified that LDRT plus chemoimmunotherapy was safe, feasible, and effective for ES-SCLC, warranting further investigation. FUNDING:This research was funded by West China Hospital (no. ZYJC21003), the National Natural Science Foundation of China (no. 82073336), and the MATCH trial was fully funded by Roche (China) Holding Ltd. (RCHL) and Shanghai Roche Pharmaceuticals Ltd. (SRPL).

Previously we have identified that the expression number and levels of oncogenes and antioncogenes are highly positively or negatively associated with major cellular progress in a cancer cell. However, we have not defined any cellular potentials of a human tumor cell at the level of the overall gene expression. Here, we counted the overall number of expression genes and overall counts of mRNA in depth and revealed that the expression levels of mRNA were directly associated with the expression number of genes in a human tumor cell. Gene expression networks revealed steady states of tricarboxylic acid (TCA) cycle and ATP production, differentiation potentials that might be disturbed and blocked by uncertain gene expressing networks, and potential capabilities to undergo epithelial-mesenchymal transition (EMT), neurogenesis, angiogenesis, inflammatory response, immune evasion, and metastasis in a human tumor cell. Our analysis identifies unpredictable gene expression characteristics in human tumor cells. The results might profoundly influence mechanisms how a human tumor cell generates and undergoes its progresses.

Vesicle trafficking is a fundamental process that allows for the sorting and transport of specific proteins (i.e., "cargoes") to different compartments of eukaryotic cells. Cargo recognition primarily occurs through coats and the associated proteins at the donor membrane. However, it remains unclear whether cargoes can also be selected at other stages of vesicle trafficking to further enhance the fidelity of the process. The WDR11FAM91A1 complex functions downstream of the clathrin-associated AP-1 complex to facilitate protein transport from endosomes to the TGN. Here, we report the cryo-EM structure of human WDR11-FAM91A1 complex. WDR11 directly and specifically recognizes a subset of acidic clusters, which we term super acidic clusters (SACs). WDR11 complex assembly and its binding to SAC-containing proteins are indispensable for the trafficking of SAC-containing proteins and proper neuronal development in zebrafish. Our studies thus uncover that cargo proteins could be recognized in a sequence-specific manner downstream of a protein coat.

Wnt/β-catenin signalling is aberrantly activated in most colorectal cancer (CRC) and is one key driver involved in the initiation and progression of CRC. However, mutations of APC gene in CRC patients retain certain activity of APC protein with decreased β-catenin signalling and DKK4 expression significantly upregulates and represses Wnt/β-catenin signalling in human CRC tissues, suggesting that a precisely modulated activation of the Wnt/β-catenin pathway is essential for CRC formation and progression. The underlying reasons why a specifically reduced degree, not a fully activating degree, of β-catenin signalling in CRC are unclear. Here, we showed that a soluble extracellular inhibitor of Wnt/β-catenin signalling, DKK4, is an independent factor for poor outcomes in CRC patients. DKK4 secreted from CRC cells inactivates β-catenin in fibroblasts to induce the formation of stress fibre-containing fibroblasts and myofibroblasts in culture conditions and in mouse CRC xenograft tissues, resulting in restricted expansion in tumour masses at primary sites and enhanced CRC metastasis in mouse models. Reduced β-catenin activity by a chemical inhibitor MSAB promoted the CRC metastasis. Our findings demonstrate why reduced β-catenin activity is needed for CRC progression and provide a mechanism by which interactions between CRC cells and stromal cells affect disease promotion.

BackgroundWhile a few case-control studies indicated a possible correlation of IgG N-glycosylation patterns with pancreatitis, their restricted sample sizes and methodologies prevented conclusive insights into causality or distinguishing traits across pancreatitis types.MethodWe conducted a two-sample Mendelian Randomization (MR) analysis to investigate the causal relationship between 77 IgG N-glycosylation traits and various types of pancreatitis, including acute pancreatitis (AP), chronic pancreatitis (CP), alcohol acute pancreatitis (AAP), and alcohol chronic pancreatitis (ACP). This analysis utilized summary-level data from genome-wide association studies (GWAS), employing methods such as IVW, MR-Egger, and weighted median. To ensure the robustness of our findings, several sensitivity analyses, including Cochran’s Q statistic, leave-one-out, MR-Egger intercept, and MR-PRESSO global test were conducted.ResultOur study uncovered the causal relationship between specific IgG N-glycosylation traits and various types of pancreatitis. Notably, an increase in genetically predicted IGP7 levels was associated with a decreased risk of developing AP. For CP, our data suggested a protective effect associated with higher levels of both IGP7 and IGP31, contrasting with increased levels of IGP27 and IGP65, which were linked to a heightened risk. Moreover, in the case of AAP, elevated IGP31 levels were causatively associated with a lower incidence, while higher IGP26 levels correlated with an increased risk for ACP.ConclusionThis study establishes causal relationship between specific IgG N-glycosylation patterns and varying risks of different pancreatitis forms, underscoring their potential as predictive biomarkers. These findings necessitate further exploration into the underlying mechanisms, promising to inform more personalized diagnostic and therapeutic strategies in pancreatitis management.

BackgroundGrowing evidence suggests that chronic inflammation, resulting from intricate immune system interactions, significantly contributes to the onset of psychiatric disorders. Observational studies have identified a link between immunoglobulin G (IgG) N-glycosylation and various psychiatric conditions, but the causality of these associations remains unclear.MethodsGenetic variants for IgG N-glycosylation traits and psychiatric disorders were obtained from published genome-wide association studies. The inverse-variance-weighted (IVW) method, MR-Egger, and weighted median were used to estimate causal effects. The Cochran’s Q test, MR-Egger intercept test, leave-one-out analyses, and MR-PRESSO global test were used for sensitivity analyses.ResultsIn the Psychiatric Genomics Consortium (PGC) database, genetically predicted IGP7 showed a protective role in schizophrenia (SCZ), major depressive disorder (MDD), and bipolar disorder (BIP), while elevated IGP34, and IGP57 increased SCZ risk. High levels of IGP21 were associated with an increased risk of post-traumatic stress disorder (PTSD), while elevated levels of IGP22 exhibited a causal association with a decreased risk of attention-deficit/hyperactivity disorder (ADHD). No causal relationship between IgG N-glycan traits and autism spectrum disorder (ASD) and no evidence of reverse causal associations was found.ConclusionHere, we demonstrate that IgG N-glycan traits have a causal relationship with psychiatric disorders, especially IGP7’s protective role, offering new insights into their pathogenesis. Our findings suggest potential strategies for predicting and intervening in psychiatric disorder risk through IgG N-glycan traits.

Embryonic axis formation is essential for patterning and morphogenesis in vertebrates and is tightly regulated by the dorsal organizer. Previously, we demonstrated that maternally derived Huluwa (Hwa) acts as a dorsal determinant, dictating axis formation by activating β-catenin signaling in zebrafish and Xenopus . However, the mechanism of activation and fine regulation of the Hwa receptor remain unclear. In a systematic mutation screen, a mutation at the 168 th serine (Ser168) in the PPNSP motif of Hwa dramatically abolished its axis-inducing activity. Mechanistically, mutating the Ser168 residue reduced its binding affinity to Tankyrase 1/2 and the degradation of the Axin protein, weakening β-catenin signaling activation. Furthermore, the Ser168 site was confirmed to be phosphorylated via LC-MS/MS and immunoblotting with a phosphorylation-specific antibody. Additionally, several kinases, including Cdk16, Cdk2, and GSK3β, were found to enhance Ser168 phosphorylation in vitro and in vivo , enhancing Hwa's activity in β-catenin signaling and axis induction. Meanwhile, the use of dominant-negative Cdk16 and pHwa (Ser168) antibodies reduced Hwa's function. In summary, Ser168 acts as a phosphorylation switch in Hwa/β-catenin signaling for embryonic axis induction, involving multiple kinases to ensure developmental robustness.

Embryonic axis formation is essential for patterning and morphogenesis in vertebrates and is tightly regulated by the dorsal organizer. Previously, we demonstrated that maternally derived Huluwa (Hwa) acts as a dorsal determinant, dictating axis formation by activating beta-catenin signaling in zebrafish and Xenopus. However, the mechanism of activation and fine regulation of the Hwa protein remains unclear. Through candidate screening we identified a mutation at Ser168 in the PPNSP motif of Hwa that dramatically abolishes its axis-inducing activity. Mechanistically, mutating the Ser168 residue reduced its binding affinity to Tankyrase 1/2 and the degradation of the Axin protein, weakening beta-catenin signaling activation. We confirmed that Ser168 is phosphorylated and that phosphorylation increases Hwa activity in beta-catenin signaling and axis induction. Several kinases including Cdk16, Cdk2, and GSK3 beta, were found to enhance Ser168 phosphorylation in vitro and in vivo. Both dominant-negative Cdk16 expression and pHwa (Ser168) antibody treatment reduce Hwa function. Lastly, a knock-in allele mutating Ser168 to alanine resulted in embryos lacking body axes, demonstrating that Ser168 is essential to axis formation. In summary, Ser168 acts as a phosphorylation switch in Hwa/beta-catenin signaling for embryonic axis induction, regulated by multiple kinases. Huluwa functions as the dorsal determinant during body axis formation in vertebrates, but how it is regulated remains unclear. Here they identify a phosphorylation switch in the conserved PPNSP motif of Huluwa required for axis induction, which is targeted by multiple kinases.

Histone modification signatures mark sites of transcriptional regulatory elements and regions of gene activation and repression. These sites vary among cell types and undergo dynamic changes during development and in diseases. Oocytes produce numerous maternal factors essential for early embryonic development, which are significantly influenced by epigenetic modifications. The profiling of epigenetic modifications during oogenesis remains uniquely challenging due to the presence of numerous tightly wrapped granulosa cells. Here, we successfully established a low-input CUT&Tag (Cleavage Under Targets and Tagmentation) method tailored for zebrafish stage I oocytes. This advanced technique enables high-resolution profiling of histone modifications and DNA-binding proteins, critical for understanding chromatin dynamics in developing oocytes. In this study, we detailed the workflow for this technique, including the isolation of pure stage I oocytes without somatic cells, library construction and quality monitoring. Our results demonstrate the method’s efficacy by identifying distinct histone modification patterns and analyzing differentially expressed genes in oocytes with and without granulosa cells. We also successfully profiled divergent histone modifications in oocytes derived from wild-type and huluwa mutants. These advancements overcome technical challenges in epigenetic research on zebrafish oocytes and establish a solid foundation for exploring the epigenetic regulatory mechanisms of maternal contribution.

Background: p73, a member of the p53 family, plays diverse roles in angiogenesis and cancer progression, likely due to the heterogeneous functions of its two primary isoforms, TAp73 and DNp73. However, its impact on the endothelial trans-differentiation of colorectal cancer (CRC) stem cells (CoCSCs) remain largely uncharted.Methods: Samples and data from the Stockholm II trial, TCGA and GEO were utilized to investigate the clinical significance of p73. We employed endothelial-specific lineage tracing of CoCSCs, RNA interference, overexpression techniques, mRNA-seq, and both in vitro and in vivo assays to delve into influence of p73 on the endothelial trans-differentiation of CoCSCs, angiogenesis and tumorigenesis. A TAp73-Induced Endothelial Differentiation Signature (TapIEDS)was constructed using 101 combinations of machine-learning algorithms and screened for potential therapeutic agents specific to high TapIEDS.Findings: Unlike DNp73, TAp73 was associated with microvascular density (MVD), and the co-expression of TAp73 and MVD served as a prognostic indicator for CRC patients. Through endothelial-specific cell lineage tracing, we further validated the endothelial trans-differentiation capability of CoCSCs. During this process, TAp73 expression was higher than that of DNp73. Silencing TAp73 inhibited the trans-differentiation of CoCSCs, while its overexpression enhanced this phenotype. However, such effects were not observed with DNp73 overexpression. Xenografts derived from CoCSCs lacking TAp73 exhibited impaired tumorigenesis and reduced vasculogenic potential, originating from both pre-existing vessels and CoCSCs-induced angiogenesis. Furthermore, TAp73 acted as a key regulator of the angiogenic transcriptome during CoCSC endothelial trans-differentiation. The TapIEDS prognostic signature, based on TAp73-regulated genes in CoCSCs endothelial trans-differentiation, was identified as an independent risk factor for patient survival. Lastly, we identified potential therapeutic agents that specifically target high TapIEDS subgroups.Interpretation: Our results emphasize TAp73 pro-angiogenic role in both pre-existing vessels and CoCSCs-induced angiogenesis, which supports tumorigenesis. The overexpression of TAp73 in CRC provides a rationale for its role in the disease and highlights promising avenues for future management strategies, including targeted therapeutic interventions.Funding: This work was supported by grants from Natural Science Foundation of Sichuan Province, China (Grant No. 2022NSFSC0764), Swedish Cancer Foundation (CAN2016/341 and 190322Pj), and Oncology Clinics in Linköping (2020–0331 and 2020–1125), and the Fundamental Research Funds for the Central Universities (Grant No.2022SCU12025 and 2022SCU12020).Declaration of Interest: No potential conflicts of interest were disclosed.Ethical Approval: This study based on patients’ data was conducted in compliance with the ethical guidelines set forth by Linköping University and was approved by the Institutional Review Board (IRB) (Approval Numbers: 2012-107-31, and 2014-79-3). All experiments involving zebrafish were conducted in compliance with the regulations and policies of the Linköping University Animal Care and Use Committee (Dnrs: 104/12, 53/14, 89/15). The study involving mice received ethical approval from the Institutional Review Board of West China Hospital of Sichuan University (Approval Number: 218156A). In accordance with the Declaration of Helsinki, all participating patients provided written informed consent prior to specimen collection.

Many cancers harbor homologous recombination defects (HRDs). A HRD is a therapeutic target that is being successfully utilized in treatment of breast/ovarian cancer via synthetic lethality. However, canonical HRD caused by BRCAness mutations do not prevail in liver cancer. Here we report a subtype of HRD caused by the perturbation of a proteasome variant (CDW19S) in hepatitis B virus-bearing (HBV-bearing) cells. This amalgamate protein complex contained the 19S proteasome decorated with CRL4WDR70 ubiquitin ligase, and assembled at broken chromatin in a PSMD4Rpn10- and ATM-MDC1-RNF8-dependent manner. CDW19S promoted DNA end processing via segregated modules that promote nuclease activities of MRE11 and EXO1. Contrarily, a proteasomal component, ADRM1Rpn13, inhibited resection and was removed by CRL4WDR70-catalyzed ubiquitination upon commitment of extensive resection. HBx interfered with ADRM1Rpn13 degradation, leading to the imposition of ADRM1Rpn13-dependent resection barrier and consequent viral HRD subtype distinguishable from that caused by BRCA1 defect. Finally, we demonstrated that viral HRD in HBV-associated hepatocellular carcinoma can be exploited to restrict tumor progression. Our work clarifies the underlying mechanism of a virus-induced HRD subtype.

Background Clinical observation suggests the atheroprotective effect of chloroquine and its derivatives, while its mechanism remains unclear. This study aimed to observe the protective effect of chloroquine against atherosclerosis and explore the underlying mechanism.Methods Ataxia telangiectasia mutated (ATM) wild-type or haploinsufficient apolipoprotein-E-knockout (ATM(+/+)ApoE(-/-) or ATM(+/-)ApoE(-/-)) mice were treated with different dosages of chloroquine. Anti-CD25 antibody was used to deplete natural Tregs in ATM(+/+)ApoE(-/-) mice. The atherosclerotic burden in different groups of mice was comprehensively evaluated by H&E staining and Masson staining. The effect of chloroquine on the regulatory T cells (Tregs) was assessed in vivo and in vitro by flow cytometry and immunohistochemical staining. The expression of related proteins was detected by real-time polymerase chain reaction and western blotting.Results In ATM(+/+)ApoE(-/-) mice, chloroquine alleviated atherosclerotic lesions, stabilized the plaque, and increased Treg counts in the atherosclerotic lesions and spleens. However, in ATM haploinsufficient mice (ATM(+/-)ApoE(-/-)), chloroquine no longer prevented atherosclerosis or impacted Treg counts. Abolishing Treg cells using an anti-CD25 antibody in vivo abrogated the atheroprotective effect of chloroquine. In vitro, chloroquine promoted the differentiation of Tregs from na & iuml;ve T cells, which was accompanied by enhanced ATM/AMP-activated protein kinase (AMPK) activity and reduced downstream mammalian target of rapamycin (mTOR) activity.Discussion These findings suggest that chloroquine ameliorates atherosclerosis and stabilizes plaque by modulating Tregs differentiation through the regulation of the ATM/AMPK/mTOR pathway.

Imaging and tracking tools for natural cellular RNA with improved biocompatibility, specificity, and sensitivity are critical to understanding RNA function and providing insights into disease therapeutics. We developed a new genetically encoded sensor using fluorogenic allosteric aptamer (FaApt) for the sensitive imaging of the localization and dynamics of RNA targets in live cells. Target RNAs can be specifically recognized with our sensor by forming perfectly complementary duplexes, which in turn can induce allosteric structural changes of the sensor to refold the native conformation of fluorogenic RNA aptamers. We demonstrated the ability of the sensor to monitor the effect of tumor necrosis factor and small-molecule inhibitor on the expression abundance of CXCL1 and survivin mRNA in human cancer cells, respectively. The asymmetrical distribution of endogenous Squint mRNA was confirmed in developing zebrafish embryos through microinjection of FaApt probes. This study provides an effective molecular tool for sensitive imaging and tracking endogenous RNA in living cells. Due to the high specificity and small size of our sensor system, it is expected to be applied to early diagnosis of RNA marker-related diseases and real-time evaluation of the treatment process.